Assessment of Clinical Chemistry Parameters and Viral Load of Hepatitis C Virus among Patients Infected with Hepatitis B Virus Attending

Hepatitis A and B are the most common causes of chronic hepatitis infections globally. This study aims at evaluating the clinical chemistry parameters of Hepatitis C virus among individuals infected with Hepatitis B virus attending Federal Teaching Hospital (FETHA), Abakaliki, Ebonyi State, Nigeria. Data such as demographic, laboratory and clinical data were collected from the hospital. Blood samples were also collected from 480 consented individuals for the screening and the presence of HBsAg was detected. Some chemistry analyses were performed to unveil the level of their immune responses using laboratory standard procedures. Of a total 480 consented patients screened, 101 individuals were reactive to hepatitis B virus surface antigen. Data obtained in our study showed an overall percentage prevalence of 45 (male patients, 24.8, and female patients 75.2). In analysis of HCV-IgM, the higher percentage was noticed among the male individuals infected with hepatitis B virus (20 %) and female subjects with 13.2 %. The IgG was higher among male subjects with 16 % and female patients with 14.5 % while patients of 52-62 years of ages had high percentage of 50 each for both IgM and IgG. Occupation was also considered as a demographic factor and farmers presented higher percentage among the occupations observed, there was a significant difference between the parameters observed in respect to their occupations (p < 0.05). Results of chemical analyses of patients positive for HCV showed higher viral load among the male counterparts and alanine aminotransferase level is higher for patients between the ages of 19-29 years. Meanwhile, increase in creatinine level was noticed among the businessmen. Conclusively, hepatitis B and C viruses are global challenges and require immediate attention.


Introduction
Hepatitis B and C viruses are viral infections that attack the organ such as the liver and therefore resulting to both acute and chronic diseases (Bullock et al., 2002). The incubation period of both viruses is 2-6 months, (60-180 days) between 15-29 years. Both viruses can result to acute and chronic stages of hepatitis, starting from severity to a mild illness which lasting for a few weeks then to a serious lifelong illness (Idoko et al., 2007).
About 250 million individuals are currently prone to viral hepatitis chronically, especially hepatitis B viruses (Surface antigen positive for hepatitis B at minimum of 6 months and hepatitis C virus globally (WHO, 2015). In some sub-Saharan Africa, about 25.4 million are now infected with this virus and 13.3 million in Nigeria (Thio et al., 2009). The prevalence of two viruses in Nigeria has been reported as 17.1 % among female sex workers (Ajuwon et al., 2016).
Occurrence of symptoms take place within four to six weeks after individuals being infected which may last from one or two weeks to several months (Abdel-Hamid et al., 2002).This signs and symptoms can include yellowing of the skin, whitening of the eyes, and under the fingernails (jaundice), dark urine or pale stool, tiredness, fatigue, fever, abdominal pain, loss of appetite, nausea, diarrhea, joint pain, vomiting and general body weakness (Cheesbrough, 2006). Both HBV and HCV share common mode of transmission, therefore, living together in the same host at significantly high rates (Idoko et al., 2007). Vertical and sexual transmission is very low, but transmission is predominantly by blood contact. The commonly used method of diagnosis of HBV and HCV viruses is antibody testing. Enzyme Linked Immunosorbent Assay (ELISA) method is useful especially during the infants testing from mothers that are positive for HCV, i.e., elevation of serum alanine transferase, serum aspartate transaminase, and bilirubin. Serology tests can also be used to detect the presence of HBV in a serum and the serological diagnosis is based on the detection of HBsAg, HBeAg, anti HBe, anti-HBs and anti-HBc and molecular analysis (Bartenschlager et al., 2003).
Treatment with antiviral medication is recommended.
Lamivudine has been extensively used in the treatment of HBV among pregnant woman in attempt to prevent prenatal transmission of hepatitis B virus infection with mixed success. Individuals with more complications should undergo treatment first because the risk of complications is determined by the degree of liver scarring (Thio et al., 2009).

Materials and Methods
Sample collection: About 5ml of blood samples were collected each from a total of 101 HBV positive patients visiting FETHA for a period of one year (2017)(2018). Serum samples were separated by low-speed centrifugation at 500 rpm for 5 minutes. Then, serum was transferred into labeled sterile cryovials and stored at -20 o C in the sterile container. The stored sample was transported to Nigerian Institute of Medical Research (NIMR), 6 Edmond Crescent, Yaba, Lagos for the analysis.

Serology Test for HCV IgM (ELISA Method)
A 200 µl of HCV-IgM was added to the negative control in triplicate (B, C and D wells) containing the antigen coated well, the blank (A well) was left without adding any diluents and it was incubated for 1 hour. A 200 µl of HCV-IgM antibodies was added to the calibrator control (E and F wells) containing the antigen coated well and 200 µl of HCV-IgM was also added to the positive control (G wells) containing the antigen coated well. A 200 µl of the sample diluents (DILSPE) was added to all the well starting from H except the controls. The colour was now observed from light green to dark bluish green. Therefore, 10 µl of the sample (plasma) was then added to all well except the controls and the blank. A50 µl of the DILAS was dispensed to all the wells including the control, calibrator except the blank and the colour turns to dark blue. It was then covered with adhesive sealing foil and the microplate was incubated for 45mins at 37 o C. After incubation, the microplate was washed for 4-5 cycles using an automated washer machine, it was empty, and the residual liquid was tapped out. A 100 µl of Enzyme conjugate was pipetted into each well except the blank and it was covered with the sealer and the microplate was then incubated again for 45 min at 37 o C. After the 2 nd incubation, the microplate was then washed for 4-5 cycles.
Serology Test for HCV-IgG (ELISA Method) Well A1 and B1 wereleft empty as blank. A 100 µl of HCV-IgG was added to the calibrators in duplicate, another 100 µl HCV-IgG was also dispensed in dissolved control serum in duplicate to the same volume of 100 µl of diluted samples. The control serum was used to verify that the whole analytical system works as expected. The microplate was incubated for 60mins at 37 o C and it was washed using an automated strip washer ELx50 for 4-5 cycles. In all the wells except A1+B1, 100 µl of enzyme conjugate was dispensed and the microplate was incubated for 60 min at 37 o C at the end of 2 nd incubation, the microwells was then washed for 4-5 cycles and a 100 µl chromogen/ substrate was pipetted into all the wells, A1+B1 included.
The microplate was protected from light by incubating it at a room temperature in a dark room usually (18-24 o C) for 20 min. Wells dispensed with positive samples, the control serum and the positive calibrators was turn from clear to blue color. At this stage, a 100 µl of Sulphuric acid was dispensed to all the wells including A1+B1 to block the enzymatic reaction. Addition of the stop solution turns the positive samples and control from blue to yellow and the color intensity of the solution in each well was measured using molecular automated Devices {(E-Max Elisa reader at 450nm filter) (Optical Density).

HCV-RNA Viral Load Test
This was done using an RNA Polymerase Chain Reaction (PCR). The plasma sample which is the best choice for viral load was used atCentre for Human Virology and Genomics (CHVG), Nigerian Institute of Medical Research, Yaba, Lagos. The samples and control were pipetted into their corresponding tubes with 3 controls for HCV-RNA assays which are negative, low positive and high positive controls. Barcoded chips were fixed into the sample racks containing the controls and the samples, both the controls and samples were placed into the racks. The sample was labelled properly, and the identification number was given to each sample.
Reagent was brought to room temperature and PCR assay conducted.

Clinical Chemistry
Liver Function Test (LFT) and Creatinine

Procedure
The machine was switched on and it was allowed to initialize for about 5 mins. The 2 controls were used which are Precinorm (Normal controls) and Precipath (Pathological controls) and the values of the controls were plotted on LJ chart, and it was accepted or rejected based on Westguard rules which are based on the performances of the control's values in the acceptance range, the patient's samples were then assayed using the equipment (Chemistry auto-analyzer, Cobas C-311). The Samples were aliquoted into sample cups, and it was placed in the groove for the samples to run, the sample ID of the patients and the corresponding test was inputted into the computer connected to the chemistry auto-analyzer machine and the command was then run and the tests was assayed.  The results of IgM screening showed that out of 25 males and 76 for females, 5(20 %) and 10(13.2 %) were positive for IgM, while for the IgG, 4(16 %) and 11(14.5 %) respectively were positive. Clinical chemistry parameters were higher in IgG than IgM, whereas IgM is higher in terms of their viral load than IgG .  The results for the IgM showed that traders had 1(10 %) for IgM, teachers, zero (0%) percent, students 6(40), farmers 3(20), businesswomen 4(26.7).

Results
In respect to the IgG, farmers had 2(13.3 %), traders 10, teachers 1(14.3), students 7(18.9). The total percentage of their clinical chemistry and their viral load was higher in IgG than IgM (Table 4).

Table 5: Clinical Chemistry Parameter of Individuals Positive for HCV-IgM and IgG in Respect to their Educational Level
The result of the IgM revealed that primary and tertiary education certificate holders gave a percentage prevalence of 5(22.7) and 5(15.2) respectively,individuals without formal education and secondary school had 2(13.3) and 3(9.7) respectively (Table 5).

Discussion.
This study provides a comprehensive sentinel surveillance of HBV and HCV among individuals infected with hepatitis B virus visiting Federal Teaching Hospital, Abakaliki, Ebonyi State. Out of 480 screened patients, 101 were positive for HBV and blood samples were collected from the positive individuals in the hospital, females examined were 380 where 76 were positive (20%) and the male subjects were 100 patients where 25 of them were positive 25 %. This is not in line with the work done by (Craxi et al., 2003 andDavis et al., 2010) which gave a percentage prevalence of 24.1 and 23.3 % respectively among students in University of Ibadan. The statistical analysis showed no significant difference among the variables in respect to the occupation (p <0.05) ( Table 4). The occupation of individuals positive for HCV-IgG showed high percentage among the farmers (33.3), out of 9 examined, 3 were positive with the ALT (16.9 µL) CRET (76 mg/dL) and viral load (3,379 mg/L). There was no significant difference between the values obtained for the viral load of HBV infected individuals positive for HCV-IgG with respect to their demographical data (p <0.05). This was in consonant with the work done by (Davis et al., 2010) (Dienstag et al., 2007). It was noticed that farmers also gave high percentage among the occupation observed in our findings. Therefore, a significant difference was noticedbetween the parameter observed in respect to their occupation (p < 0.05). The level of education of an individual's results revealed that primary school certificate holder showed higher percentage whereas higher percentage of ALT were found among the farmers, this signified the level of liver damage (Gay et al., 2001). This research provided opportunity to create awareness especially on hepatitis B virus where majority leaving in the rural area were not informed. Therefore, government should structure some health policies that will involve both HIV and HBV as a routine screening for antenatal patients visiting health care facilities in Nigeria. Both the acute and chronic states of hepatitis B and C treatments must begin immediately after positive diagnosis.
viruses can result to acute and chronic stages of hepatitis, starting from severity to a mild illness which lasting for a few weeks then to a serious lifelong illness. Jou. of herpetolo,